1. Field of the Invention
The invention relates to a process for detection of analytes and a device for carrying out the process.
Such devices and processes, which are also designated below with the term of assay describing both, serve for qualitative and quantitative recording of specific bonds between at least two molecules. This includes, for example the recording of receptor-ligand interactions, antibody-antigen interaction, recognition of nucleic acids, interaction between oligonucleotides and DNA as well as other molecular interactions. Processes and devices of this type can be used, for example in chemistry, clinical analysis, pharmaceutical development, environmental analysis and in routine molecular biology work including sequencing of nucleic acids.
It is known that immunoassays having different detection methods are carried out. This includes radioactive, fluorescence-assisted and chemoluminescence-assisted and enzymatic processes (C. P. Prince, D. J. Newman: Principles and Practice of Immunoassays, Macmillan Publishers Ltd., 1991 U.K.).
In a particular form of immunoassay there is an agglutination of latex particles due to antibody-antigen bonds, which may be detected, for example visually (J. M. Singer, C. M. Plotz: The Latex Fixation Test, American Journal of Medicine, December 1965, pp. 888-892). 105 molecules may be detected using microspheres of 10 xcexcm diameter, 108 molecules using microspheres having a diameter of 1 xcexcm, and 1013 molecules using microspheres having a diameter of 0.1 xcexcm with such agglutination tests. Using the example of IgG (MW=100,000) theoretical sensitivities of 10 fM, 10 pM or 10 nM are given. The highest sensitivity is thus seen for relatively large microspheres, the use of which is however limited by their sedimentation behaviour,
Furthermore, recently nucleic acids, for example oligonucleotides, RNA and DNA may be detected via such interactions by means of DNA microchip technology (Nature, Genetics, Volume 14, No 4, December 96, and D. Noble: DNA Sequencing on a chip, Anal Chemistry, Volume 67, No. 5. Mar. 1, 1995). However, the chip technology is not used here as an electrical measuring process, but it serves as a novel synthesis process and for producing microstructures. The actual detection mechanism is of visual type. The combination of electrical processes for synthesis of a ligand and the visual marking and detection is however very expensive.
The disadvantage of the state of the art is that detection processes based on radioactivity are burdened with problems of radiation protection and disposal of the radioactive waste thus produced. In enzymatic detection methods which facilitate electrochemical detection of the analytes, a chemical reaction with a substance as the chemical reaction substrate must take place as an additional working step.
In all detection processes and assays known in the state of the art, a final washing step is necessary to remove excess reactants before detection of the analyte to minimise nonspecific signals as far as possible.
The object of the invention is to provide a device and a process which facilitates detection of analytes in rapid, simple and precise manner and in which a washing step may be omitted.
2. Description of the Related Art
World application 9005300 relates to a process for detection of binding reaction between specific substances, in which electrodes, between which a gap exists, are applied to a support. A layer of, for example antigens, is applied to the support in the gap. A measuring solution containing marker particles with antibodies and antigens is passed over the support, wherein marker particles with antibodies bind to the antigen layer of the support. After a preset reaction time, the non-bound marker particles are removed by washing and after drying the resistance is measured between the two electrodes which is a measure for binding which has taken place. Depending on the design of the support (conducting or non-conducting), a signal is measured when the gap is not completely bridged by conductive particles or only when the gap is filled.
European application 0 745 843 discloses an electrochemical biosensor which has electrodes and a matrix with different reagents. The reagents include conjugates of analytes and cytolytic reagent, receptors and liposomes containing electroactive materials. The liposomes release the electroactive material under the action of the analyte.
In a biosensor disclosed in European application 0 402 917, an electrically conducting polymer layer, to which a binding partner is coupled, is arranged between the electrodes. When binding the particular binding partner, the electrical properties of the polymer layer are changed.
European application 0 495 519 describes a process for measuring a specific substance in a sample, wherein an alternating current is applied to electrodes shaped like ridges to accelerate the aggregration of charged particles. Aggregation is then measured visually.
This object is achieved according to the invention by the features of the main and sub-claim.
If marker particles having electrical and/or electrochemical properties, which differ from those of their surroundings, the measuring solution, are introduced into an electric field, the electric field is disturbed by this. These disturbances of an electric field produced in the measuring solution may be determined simply, rapidly and very precisely.
The process of the invention is thus based on a detection method which may be regarded as essentially purely electrical. Therefore it has the very high sensitivity or precision possible for the determination of electrical or electrochemical properties and consequently a very low detection limit at high sensitivity.
With suitable dimensions and/or positioning of the electrodes which produce the electric field, the electric field is virtually exclusively influenced by marker particles having different material composition, different specific resistance of electric surface charge or different dielectric constant, which have undergone specific binding, for example with the analyte or with a substrate, that is bodies or materials suitable as substrate. Excess non-bound marker particles do not lead to a signal, so that a washing step for removing non-bound marker particles from the measuring solution may be omitted.
The measuring range of the bioassay according to the invention (process and/or device) may be adjusted by fixing the electrode or substrate surfaces and by selecting the size of the marker particles.
The process of the invention and the device of the invention may be used for detection and for concentration determination of any analytes which can be recorded via molecular interactions. These include, for example the interactions of receptor-ligand, antibody-antigen, antibody-hapten, antibody fragment-antigen, aptamers, proteins, nucleic acids, oligonucleotides, DNA and all molecular interactions, in which at least one of the molecular partners may be marked with a marker particle. This includes the interaction of materials with the surfaces of whole cells. It is possible in principle to realise all known immunoassay formats according to the state of the art.
The advantages achieved using the invention consequently exist particularly in the fact that a purely electrical detection method is used having all its advantages with reference to precision, rapidity and sensitivity, and that also when using very small electrodes and marker particles of comparable size, individual bond detection is possible.
Advantageous further developments of the process of the invention and the device of the invention are given in the dependent claims.
Particularly in the near-field of an electrode producing an electric field, even the presence of a single marker particle may lead to adequately high changes in the electric field. Non-specifically bound marker particles further removed from the electrode lead to fewer impairments of the electric field, so that with suitable arrangement of the electrodes in the vicinity of a substrate or design of the electrodes themselves as a substrate and at suitable concentration of the marker particles, the measured signal is influenced essentially only by specifically bound marker particles. Hence, a washing step for removing excess marker particles or analytes from the measuring solution may then also be omitted.
When using marker particles, the relative permeability of which differs from the relative permeability of the measuring solution, wherein a magnetic field is produced at or in the measuring solution, detection may be provided by the strength of the magnetic field or by its change caused by the marker particles.
Hence, for example individual bond detection may take place at an electrode as substrate, the surface of which lies in the same order of magnitude as the largest cross-sectional surface area of the marker particle or at least does not differ by several orders of magnitude. Hence microelectrodes may be used for this, which have round, square, rectangular, but also any shape. The diameters of the marker particles lie between a few nm to a few xcexcm, for example 10 xcexcm.
If binding of several marker particles is to be detected by measuring technology, larger electrodes may be used, at the surface of which there is surface covering including three-dimensional agglutination of marker particles. In contrast to the values given for the state of the art for agglutination tests, the theoretical detection limit drops as a function of the diameter of the marker particles by several orders of magnitude.
Furthermore, the measuring range of the bioassay may be adjusted by fixing the electrode surfaces between the individual bond region and the agglutination region.
When using only one microelectrode or very few microelectrodes (with an associated counter-electrode), very low analyte concentrations may be recorded when the measuring medium surrounding the microelectrode(s) has just a small volume with a limited number of marker particles. Simpler, more precise individual bond detection may therefore be realised by using a very small sample chamber or through-flow chamber in the order of magnitude of xcexcl.
This applies particularly in the detection of analytes by their specific binding to marker particles in a through-flow measuring system, wherein here the analyte stream takes with it the bound marker particles by means of an electric field applied externally via electrodes. The changes in electric field which are shown as changes in the electric current or capacity between the electrodes in the measuring solution and which are triggered by the marker particles, may be recorded precisely. Individual detection is also possible here with a corresponding low volume of the through-flow region.
If an electric or magnetic field alternating its polarity is applied to the measuring solution, improved mixing of the analytes and the marker particles may be achieved by the movement thus caused of electrically charged or magnetic (paramagnetic or diamagnetic) marker particles. Paramagnetic marker particles are particularly suitable for this, since a considerably lower magnetic field is required than for diamagnetic marker particles. The precision and reproducibility of any marker-assisted detection process may be improved by such field-induced mixing. The marker particles having immobilised molecules may thus also include those which do not carry any molecules. These additional marker particles increase the mixing effect. This mixing of the measuring medium may additionally be supported by connecting ultrasound.
Furthermore, such marker particles may be moved to their binding sites on the substrate by corresponding electric or magnetic fields due to electrophoretic or magnetically induced transport, or may be removed from the vicinity of the substrate after binding of excess marker particles is completed. The markers present in the measuring solution are better utilised due to this electrophoretic or magnetic field-induced transport of the marker particles to their binding sites, and the sensitivity, the detection limit and the reproducibility as well as the precision of the processes of the invention is greatly improved. Due to the electrophoretic or magnetic field-induced transport of the residual non-bound marker particles, taking place on the bond of the marker particles at their binding sites, away from the binding sites, their concentration is greatly reduced in the region of the substrate and/or the electrodes. As a result of the sensitivity of the electrodes to disturbances, particularly in their near-field, the free marker particles still influence the measurement only insignificantly. A special washing step, which is necessary according to the state of the art for many analytical processes, in particular immunoassays, may also therefore be omitted.
If the molecules to be detected in the measuring process and the molecule-charged marker particles have electric charges of different polarity, when applying an electric voltage, electrophoretic transport initially transports the charged molecules to their binding sites on or in the surroundings of the electrodes. After binding to the binding sites is completed, by reversing the polarity of the electric field, the molecule-charged marker particles are brought to the binding sites on or in the vicinity of the electrodes. This process is particularly advantageous when using sandwich format assays.
The described direct transport of electrically charged or magnetic particles induced by an electric or magnetic field and alternating transport orientated to mixing, can be used for all marker-assisted detection processes proceeding in measuring solutions.
When using inhomogeneous electric fields, uncharged, but polar marker particles may also be transported directed by the process given above or mixed.
In accordance with an exemplary embodiment of the invention, an electrode is used in front of which a diaphragm having at least one small opening is arranged. The diaphragm thus faces the measuring solution and via the electrode an inhomogeneous electric field is produced, the field lines of which pass through the small opening in the diaphragm. The marker particles are bound to or in the vicinity of the surface of the diaphragm. The advantages achieved using this embodiment consist particularly in that to produce an inhomogeneous electric field, a microelectrode having a diameter in the xcexcm range and smaller does not have to be produced, since the diaphragm opening is important for the electric field. The production of microelectrodes is indeed known, but it requires an expensive technological process with high costs. When using a diaphragm in front of a macroelectrode, the technological requirements in electrode production are not so high, so that the device of the invention can be produced with low costs. Hence, measuring devices with individual electrodes or electrode arrays may be produced as disposable articles with low costs.
A further advantage of the invention consists in that the current which flows through the small diaphragm opening to the counter-electrode in the measuring solution, only causes a very small current density at the electrode. Compared to using a microelectrode, in this case the current density is smaller by the ratio of the cross-sectional surface area of the diaphragm opening and electrode surface. This results in changes in the electrical resistance being caused mainly by particle-induced field disturbances and the influence of electrochemical electrode reactions is largely negligible. The measurements may be carried out more precisely in this manner.
In accordance with a further exemplary embodiment of the present invention, the measurement of detection of the disturbances to the electric field caused by the marker particles is carried out in the measuring solution using a potentiometric process, wherein the electric field is formed at the surface of an electrode by potential-forming steps at the boundary between measuring solution and electrode.
The advantages of this embodiment consist particularly in that likewise electrodes having greater diameter or greater surface may be used. This can be seen from the fact that the potential-forming steps on the surface of a potentiometric electrode occur at distances from this electrode which lie in the same order of magnitude as the diameter of marker particles. Marker particles in the nm range to the xcexcm range may be used in this manner.
It is important for application in a potentiometric measuring process that the marker particles have a significantly different electric charge and/or potential difference in the liquid measuring medium at their surface than the ion-selective electrode itself. This is a favourable premise in potentiometric detection for binding of marker particles.
It is additionally possible both for the amperometric and for the potentiometric detection process to influence the potential ratios at the surface of the marker particles in that the marker particles are doped with ionophores, as is also the case for ion-selective membranes. Other ionophores should thus be used than in the ion-selective membrane which serves for detection of the bound marker particles. Likewise, it is possible to provide marker particles with a thin metal layer on the surface.
Both in the amperometric and in the potentiometric detection process, detection of analytes may be effected in a two-step process which comprises marker particle transport and then binding to the electrode, As described, electrophoretic and magnetic marker particle transport may be used here.
Exemplary embodiments of the invention are shown in the drawing and are illustrated in more detail in the description below.